- Purpose:
Transforming growth factor-beta (TGF-beta) is a potent anti-mitogenic factor for many different cell types. TGF-beta initiates signaling through activation of specific TGF-beta type I and type II receptors (TbetaR-I and TbetaR-II, respectively). Whereas Smad2, Smad3 and Smad4 have been identified as pivotal intracellular effectors for TGF-beta, there is growing evidence that other (parallel) pathways exist, e.g. small GTP-binding proteins as well as MAP kinases, are activated by TGF-beta in certain cell types. Escape from TGF-beta-induced growth arrest, e.g. through functional inactivation of Smad2 or Smad4, is frequently observed in particular tumor subsets. However, such cells may have retained certain TGF-beta-induced responses as TGF-beta actively promotes tumor cell growth, invasion and metastasis during late phases of tumorigenesis. The specific objectives of the proposed study are: (1) to identify TGF-beta signaling pathways that are distinct from Smad pathway, (2) characterize their functional importance and relationship, if any, to the Smad pathway and (3) to determine the effect of inactivating or redirecting the TGF-beta/Smad pathway on TGF-beta-induced cellular responses.
- Plan of investigation:
We will investigate in two ways the activation of TGF-beta intracellular components distinct from (particular) Smad proteins: (1) by elucidating TGF-beta mediated responses that are initiated from a TbetaR-I mutant defective in Smad activation, and (2) by determining TGF-beta-induced effects on cells that are deficient in Smad3 or Smad4.
1. We have made a TbetaR-I with a mutation in the L45 loop of the kinase domain, termed TbetaR-I/D266A, with retained kinase activity that is unable to activate Smads, but capable of MAP kinase activation. We will investigate which signaling pathways are activated by TGF-beta in TbetaR-I/D266A transfected epithelial and endothelial cells and fibroblasts that lack endogenous TbetaR-I. The relationship of these activated signaling pathways with the Smad pathway will be investigated using TGF-beta-induced Smad phosphorylation, nuclear accumulation and transcriptional activation as read-outs. In addition, mock and TbetaR-I wild-type or TbetaR-I/D266A stably transfected cells will be analyzed with respect TGF-beta-induced actin reorganization, small G-protein activation, transwell migration in Boyden chambers and migration into three-dimensional collagen gels. In addition, we will measure TGF-beta-induced effects on gene expression using cDNA microarrays on RNA isolated from cells deficient in TbetaR-I in the absence or presence of ectopic expressed TbetaR-I/D266A. Furthermore, we will investigate (together with another research group) the in vivo significance of receptor-initiated responses distinct from Smads by generating flies expressing a type I receptor with mutation in L45 loop defective in Smad activation.
2. Smad3 or Smad4 deficient cells were established from Smad knock-out mice. MDA-MB 468 breast carcinoma cells have a homozygous deletion for Smad4. We will identify genes/proteins whose expression is altered in these cells upon TGF-beta challenge using cDNA micro-arrays and/or proteomics. A few selected TGF-beta target genes/proteins from both approaches will be further characterized with respect their significance in various TGF-beta responses, including effects on migration and epithelial-mesenchymal transition, using standard biochemical approaches, including overexpression of constitutively active or dominant negative forms, anti-sense techniques or inhibition with low molecular weight inhibitors.
- Possible results and relevance for cancer research:
During late phases of tumorigenesis TGF-beta has been shown to act as a tumor promoter in a cell-autonomous manner. Elucidating the molecular and cellular events that occur upon blunting or redirecting the TGF-beta/Smad pathway leading to growth arrest will provide new insights into the pathological role of TGF-beta in carcinogenesis. The identified targets that become activated independent of (particular) Smad proteins are potential targets for development of novel therapeutic strategies to treat cancer patients and may have diagnostic value. |
